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Search for the candidate gene of a novel polyglutamine disease using proteomics.

利用蛋白质组学寻找新型多聚谷氨酰胺疾病的候选基因。

基本信息

批准号：
17500225
负责人：
TOYOSHIMA Yasuko
金额：
$ 2.24万
依托单位：
Niigata University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

项目摘要

We have found three novel polyglutamine (polyQ) disease families in last four years. In histological examination of the nervous system, each case has shown unique distribution of the polyQpositive neuronal nuclei. We revealed one case was a homozygote of SCA17, and reported the clinico-pathological findings.After informed consent, we analyzed the protein extracted from autopsied brain. We found a case had an extra band in western blotting pattern using antibody to polyQ stretches (1C2).To profile the expression of proteins, we used 2D fluorescence difference gel electrophoresis (2D-DIGE) system (Amersham Bioscience). We chose a patient, who had been revealed as novel polyQ disease, and six controls. The protein samples were extracted from their cerebellum, and were labeled with CyDyes (patient : Cy5, control : Cy3, and internal standard : Cy2). The samples were separated over first and second dimensions according to their charge and size, respectively. Once the samples had been separated in the second dimension, gels were scanned for Cy2, Cy3 and Cy5 fluorescence using an appropriate scanner, Typhoon TM 9400 imager (Amersham Bioscience). And image analysis was performed using DeCyder (Amersham Bioscience). As a result, we discovered a certain protein which was expressed massively in the patient's brain. Besides, from the result of 2D western blotting, the protein was the very thing that we have recognized as an extra band in the 1D western blotting with 1C2. We picked the protein spot, and sequenced the peptide fragments by MALDI-TOF mass spectrometry. We could not find the candidate gene, however, we could build up the systems to find abnormal proteins quickly.

近四年来，我们发现了3个新的聚谷氨酰胺病家族。在神经系统的组织学检查中，每个病例都显示出独特的多q阳性神经元核分布。我们发现1例为SCA17纯合子，并报道了临床病理结果。在知情同意后，我们分析了从尸体解剖的大脑中提取的蛋白质。我们发现一个病例在polyQ延伸抗体（1C2）的western blotting模式中有一个额外的条带。为了分析蛋白质的表达，我们使用了2D荧光差凝胶电泳（2D- dige）系统（Amersham Bioscience）。我们选择了一名被发现为新型多q疾病的患者和六名对照组。从他们的小脑中提取蛋白质样品，用cydye标记（患者：Cy5，对照组：Cy3，内标：Cy2）。根据样品的电荷和大小分别在一、二维上进行分离。样品在第二次元上分离后，使用合适的扫描仪Typhoon TM 9400成像仪（Amersham Bioscience）对凝胶进行Cy2、Cy3和Cy5荧光扫描。使用DeCyder （Amersham Bioscience）进行图像分析。结果，我们发现了一种在病人大脑中大量表达的蛋白质。此外，从2D western blotting的结果来看，该蛋白正是我们在1C2的1D western blotting中识别出的额外条带。选取蛋白位点，利用MALDI-TOF质谱法对肽段进行测序。我们无法找到候选基因，但是，我们可以建立快速发现异常蛋白质的系统。